Integra modelo regeneração dérmica foi gentilmente cedido pela Integra Life Sciences Corporation. Fontes de fundos de apoio à obra: Este trabalho foi parcialmente financiado pela CIRM-BMBF precoce Translational II Prêmio e do Centro FONDAP para a regulação do genoma, tanto para JTE (Nr 15090007.).

1 Preparação de andaimes <ol><li> Gerar amostras dos scaffolds usando 12 milímetros punções. </li><li> Para introduzir moléculas bioativas ou células para o cadafalso, drenar os andaimes delicadamente apertando-os com uma gaze estéril. Em seguida, re-hidratar os andaimes por adição de 160 ul de uma solução contendo as moléculas bioactivas ou células de interesse. Confira o sucesso de bioactivação com células através de ensaios metabólicos, como MTT Ensaios. </li><li> Se necessário, corrigir os compostos ou as células a serem testadas dentro do andaime, fornecendo-lhes, por exemplo, em uma solução de fibrina-trombina ou um hidrogel, de novo por meio de re-hidratação, conforme descrito no passo 1.2. NOTA: Este passo pode ser útil quando os compostos ou as células não dão mecanicamente com o material. Pode-se também controlar a dinâmica de libertação dos compostos .. </li><li> Prepare as malhas titanizada, que serão colocados sob cada andaime em cada grupo experimental e controle, por cortandog pedaços em forma redonda, de aproximadamente 14 mm de diâmetro. NOTA: A malha titanizada funciona como uma fronteira física necessária para delimitar o tecido regenerado a partir do leito da ferida. </li></ol> 2. Animais <ol><li> Antes da implementação do modelo apresentado, consultar as leis de proteção aos animais correspondentes e obter a permissão das autoridades locais. Neste trabalho, todos os experimentos foram realizados com camundongos, de acordo com a atual lei de protecção dos animais alemão e aprovado pelo Governo do Distrito da Alta Baviera (Regierung von Oberbayern). </li><li> Escolha cuidadosamente o animal e tensão. Apesar de se ter estabelecido o modelo de ratos, que podem ser convertidos para outros animais comuns, tais como ratos, coelhos ou porcos. </li><li> No caso de você estiver trabalhando com animais de pêlo, raspar a parte de trás dos animais antes da implantação dos andaimes. NOTA: Este trabalho utiliza ratos de 6 a 8 semanas de idade, com peso corporal entre 20 ge 25 g, no entanto diferentes idades e bpesos ody também pode ser usado. </li></ol> 3 Anestesia <ol><li> Conduzir a operação em condições estéreis. A fim de manter a temperatura normal do corpo do animal, utilizar um tapete de aquecimento. </li><li> Antes da anestesia por inalação os animais recebem buprenorfina a uma dose de 0,05-0,1 mg / kg de peso corporal, por via subcutânea a cada 8 horas. </li><li> Colocar o animal sob anestesia inalação (isoflurano) ou aplicar procedimentos padrões equivalentes. Confirme anestesia ou apertando suavemente os dedos do animal ou através de uma pitada de pele. </li><li> Para evitar exsiccosis, injectar 0,5 ml de solução salina fisiológica por via subcutânea, no início da operação. </li></ol> 4. Excisão da Pele <ol><li> Colocar o animal em posição prona e marcar a linha média das costas do rato com uma ponta fina caneta permanente (Figura 1A). </li><li> Definir a área da excisão. Coloque os defeitos na área mostrada na Figure 1C. Note-se que se os defeitos são colocados muito caudal, os animais são mais propensas a remover os curativos e os andaimes. </li><li> Criar rodada defeitos bilaterais através de um 10 milímetros biópsia (Figura 1B). O perfurador deve ser cuidadosamente forçado contra a pele e não deve ser usada para cortar completamente através da pele, mas para delinear a área de excisão (Figuras 1C e 1E). </li><li> Delicadamente levante a pele marcada com uma pinça e inciso ao longo do círculo marcado com tesouras cirúrgicas finas (Figura 1F). Em caso de hemorragia, comprimir cuidadosamente com gaze estéril. Uma descrição passo a passo da excisão é mostrado na Figura 1. NOTA: O defeito é criado com um perfurador menor do que aquele para gerar os andaimes, para compensar o alargamento do defeito devido à elasticidade da pele. </li></ol> 5. andaime Implantação <ol><li> A fim de abrir espaço para a titazado malha, estender a separação da pele e do tecido subjacente nas fronteiras da ferida para 2-4 mm (Figura 2A). </li><li> Coloque a titanizada engrenar no defeito directamente no leito da ferida e sob as bordas da ferida (Figuras 2A e B). </li><li> Coloque andaimes diretamente sobre a malha. </li><li> Suturar os andaimes para as bordas da ferida adjacente por 4 a 6 nós individuais, deixando os bordos ligeiramente ao longo do esqueleto (Figura 2C). NOTA: Evite o uso de suturas biodegradáveis. </li><li> Suturar uma ferida curativo transparente acima dos defeitos para proteger a armação, enquanto que permite um controlo da área da ferida (Figura 2D). </li></ol> 6 Cuidados Pós-Operatórios <ol><li> Mantenha um rato por gaiola para evitar manipulação mútua do curativo e andaimes. </li><li> Avaliar o estado geral do animal diariamente pela atividade observando motor, peso do corpo, sinais de dor, tolerânciaao molho e auto-mutilação. Também monitorizar a área da ferida para o sangramento, local e sintomas sistémicos de infecção e a posição do penso. </li><li> A fim de minimizar a dor do animal, injecta-buprenorfina por dia numa dose de 0.05-0.1 mg / kg de peso corporal ou analgésicos equivalente. </li><li> Se o rato elimina ou danifica o curativo, substituir ou recolocá-la sob anestesia. </li></ol> 7 Eutanásia e explantes do andaime <ol><li> Em intervalos de tempo desejados, sacrificar os animais por overdose de pentobarbital (150 mg / kg). </li><li> Marque o local da excisão de pele com um marcador permanente, que deve incluir os andaimes e tanto o mouse de volta pele possível (Figura 3A). </li><li> Faça uma incisão na pele ao longo das linhas marcadas com uma tesoura ou um bisturi. Retire toda a pele, incluindo os andaimes e malha titanizada, do tecido subjacente através de uma preparação sem corte (Fifigura 3B). </li><li> Colocar o tecido explantado estendido sobre uma placa de Petri (Figura 3C e D) </li></ol> 8 visualização e quantificação da Rede Vascular <ol><li> Transiluminação: <ol><li> Configurar um dispositivo transillumination, com a montagem de uma placa de Petri sobre uma forte fonte de luz branca (100 Watt lâmpada padrão). </li><li> Corrigir uma câmera digital acima da transilluminator obter fotos digitais. </li><li> Colocar as amostras de cabeça para baixo na placa de Petri sobre o dispositivo transillumination. </li><li> Tirar fotos dos andaimes completos em modo macro, bem como de áreas de igual tamanho de pele normal. Armazenar as imagens em um formato TIFF, para posterior análise digital. </li><li> Salve o tecido para posterior análise bioquímica ou histológica. </li></ol></li><li> Segmentação Digital e quantificação: <ol><li> Baixe e instale o software VesSeg-Tool, que pode ser obtido free de carga em: <a href="http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255">http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255</a> . </li><li> Abra a imagem com o software VesSeg-Tool. </li><li> Selecione a área da imagem coberta pelo andaime para análise digital (Imagem → Select). </li><li> Para aumentar o contraste dos vasos use a opção &quot;thresholding histerese&quot; (Imagem → melhoria navio filtro → histerese limiar → Top-Hat transformação → melhoria navio Calcular). </li><li> Prossiga com a segmentação do mapa navio (fronteiras Imagem → melhoria navio filtro → histerese limiar → limiar). Selecione o primeiro limiar (&quot;cobertura de navio&quot;), de modo que cada pixel, o que é mesmo remotamente navio-like, serão rotulados como vaso. Selecione o segundo limiar (&quot;cobertura de Fundo&quot;), para que apenas os pixels que são particularmente navio-como vai ser rotulado como vasos. </li><li> Confira a proposta de segmentação automática manualmente para adicionar navios não capturados e eliminar estruturas falso-positivos comuns, que geralmente são estruturas longas e finas de navios-like, como cabelos ou suturas. </li><li> Calcule o comprimento dos vasos e a área total coberta por navios (estatísticas Imagem → Imagem → estatísticas imagem binária). NOTA: Para o cálculo dos comprimentos de navios, navios no mapa navio resultando sejam reduzidas a linhas com uma largura de um pixel 20. </li></ol></li><li> Analisar a área da pele nativa sob os mesmos parâmetros que os andaimes, a atribuição de um valor de 100% para o tecido nativo e relacionar o andaime para esse valor. Por exemplo, se a percentagem de pixels brancos é de 60% no tecido normal e de 30% no andaime, que representaria uma vascularização 50% do andaime. Nota: A cobertura branca vasos é atribuída à área de um quadrado em torno do andaime. Ajuste o número de pixels brancos consi coberturadering que a área do círculo é significativamente menor (π x R 2) do que o quadrado (4 x R 2). </li></ol>

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Declaração de conflito de interesse: Todos os autores: Nenhum Divulgações financeiras: Nenhum dos autores tem interesse financeiro em qualquer um dos produtos, dispositivos ou drogas mencionadas neste artigo.

Há uma necessidade de se estabelecer abordagens bem sucedidas na melhoria da perfusão sanguínea em engenharia de tecidos construções, o que exige o desenvolvimento de novos métodos confiáveis ​​para estudar os processos de vascularização dentro dos biomateriais. Os métodos mais comuns para fazer andaime vascularização ex vivo disponíveis incluem a utilização de microscopia, que proporciona uma ferramenta de alta-resolução. Na maioria dos casos, no entanto, este método é limitado à análi...

Em décadas recentes, a engenharia de tecidos, abriu uma nova opção terapêutica para substituir defeitos de tecidos com células do próprio corpo um. A fim de apoiar o processo fisiológico da regeneração de tecidos, andaimes são concebidos como uma estrutura biodegradável, que proporciona um cenário em que as células do leito da ferida pode crescer e restaurar o defeito de 2,3. Vascularização insuficiente é considerada o principal obstáculo que impede o avanço clínico de andaimes Bioartificiais 4. Com o crescimento de células, a procura de nutrientes e de oxigénio e aumento da vascularização do material torna-se essencial. Vascularização insuficiente ou atrasada pode, portanto, levar à necrose central de produtos da engenharia de tecidos 5. Além disso, os vasos sanguíneos fornecem células imunocompetentes e remover os resíduos metabólicos na zona de regeneração. Altas taxas de infecção e baixa regeneração são apenasalgumas das consequências de perfusão sanguínea insuficiente observados na engenharia de tecidos, que são destinadas a ser evitada pelo aumento da vascularização dos andaimes 6,7. Várias estratégias que visam melhorar a vascularização foco no papel fundamental da própria biomaterial ea microestrutura do andaime. Há esforços de pesquisa intensiva para desenvolver novas abordagens em mudar o processo de cicatrização de reparação para a regeneração, assim, (re) produzir um tecido com propriedades fisiológicas mais próximos o único a ser restaurados 8,9. Biomateriais que foram estudados e avaliados no que diz respeito ao seu potencial regenerativo incluído colágeno, fibrina, quitosana e alginato 10,11. Estes biomateriais podem ser usados ​​e combinados como uma espinha dorsal para a construção de novos scaffolds utilizando diferentes estratégias como descelulação tecido, auto-montagem, prototipagem rápida e eletrofiação 12. A fim de ENHance própria capacidade regenerativa do corpo, andaimes podem ser bioactivado. A incorporação de crescimento angiogénico recombinante factores 13 ou genes vectores que codificam para factores tais 14 demonstrou melhorar a vascularização do andaime. A utilização de células estaminais tem sido amplamente demonstrado ser uma estratégia promissora para melhorar a vascularização, em que as células estromais mesenquimatosas e células progenitoras endoteliais ganharam mais atenção 15,16. Outras abordagens tentam construir estruturas que contêm redes de vasos pré-fabricadas antes do transplante 17. Apesar dos esforços intensivos em design de andaime e sua bio-ativação, nenhuma estratégia melhorou a vascularização em um nível clinicamente significativo e, com exceção de substituições dérmicas em queimaduras maciças, a tradução de materiais de bioengenharia na rotina clínica é só ocorrendo hesitante 18 . Uma das razões pelas quais a vascularizaçãode construções de tecidos artificiais é ainda um problema não resolvido, é a dificuldade de avaliar o sucesso das novas tecnologias em abordagens in vivo. Embora experiências in vitro pode fornecer informações importantes sobre o potencial de vascularização de andaimes, modelos animais apropriados são necessários para estudar os parâmetros-chave, tais como a biocompatibilidade do material, a segurança ea eficácia do tratamento e, de particular importância, a vascularização do tecido construir. Portanto, ferramentas confiáveis ​​para visualizar e quantificar redes de vasos sanguíneos in vivo são essenciais. Neste estudo apresentamos um método simples e confiável que permite a visualização e quantificação da rede vascular no interior andaimes explante. Este método baseia-se no tecido de transiluminação e segmentação digital. Uma vez que este método é não-invasivo, que permite novas análises moleculares e histológicos do material alvo.

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			<td height="40" style="height:40px;">Gazin Mullkompresse&#160;</td>
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			<td>Sterile gauze (10 cm x 10 cm)</td>
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			<td height="40" style="height:40px;">Double-layer collagen-based scaffold (8 &#39;x 10&#39;)</td>
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			<td height="40" style="height:40px;width:319px;">Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu</td>
			<td style="width:223px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:119px;">Strain code 088</td>
			<td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&#160;</td>
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			<td height="40" style="height:40px;width:319px;">Buprenorphine (0.3 mg/ml)</td>
			<td style="width:223px;">Essex Pharma GmbH, Munich, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="40" style="height:40px;">Titanized mesh (15 cm x 15 cm), extralight</td>
			<td style="width:223px;">PFM Medical AG, K&#246;ln, Germany</td>
			<td style="width:119px;">6000029</td>
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			<td height="40" style="height:40px;width:319px;">Tissucol Duo S Immuno 2 ml</td>
			<td style="width:223px;">Baxter Germany GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;">B1332020110614</td>
			<td>Fibrin-thrombin solution&#160;</td>
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			<td height="40" style="height:40px;">Transparent adhesove drape (30.5 cm x 26 cm)</td>
			<td style="width:223px;">KCI Medical Products, Wimborne Dorset, UK</td>
			<td style="width:119px;">M6275009/10</td>
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a full skin defect model to evaluate vascularization of biomaterials in vivo

Vascularização insuficiente é considerada um dos principais fatores que limitam o sucesso clínico de construções de engenharia de tecidos. A fim de avaliar novas estratégias que visam melhorar a vascularização, métodos confiáveis ​​são necessários para fazer o em-crescimento de novos vasos sanguíneos em andaimes bio-artificial visíveis e quantificar os resultados. Ao longo dos últimos dois anos, nosso grupo apresentou um modelo de defeito pele completa que permite a visualização direta dos vasos sanguíneos por transiluminação e oferece a possibilidade de quantificação através da segmentação digital. Neste modelo, uma cria cirurgicamente defeitos da pele cheio nas costas de camundongos e substitui-los com o material testado. Moléculas ou células de interesse, também pode ser incorporado em tais materiais para estudar o seu efeito potencial. Depois de um tempo de observação de uma de própria escolha, os materiais são explantada para avaliação. Feridas bilaterais oferecem a possibilidade de fazer comparações internas ªa minimizar artefactos entre indivíduos, bem como de diminuir o número de animais necessários para este estudo. Em comparação com outras abordagens, o nosso método oferece uma análise simples e eficaz, confiável e de custo. Nós implementamos este modelo como uma ferramenta de rotina para realizar a triagem de alta resolução ao testar a vascularização de diferentes biomateriais e abordagens bio-ativação.

Um defeito da pele completa bilateral de confiança podem ser criados no ratinho (Figura 1), em que a pele pode ser substituído por um biomaterial sob estudo (Figura 2). Aqui, sem grandes complicações são observadas durante ou após o procedimento operatório, nem sinais macroscópicos de infecção ou reação de corpo estranho. Em casos raros, um andaime se perde quando um rato remove-lo. Contracção da ferida não foi observado (Figura 3). Transiluminação teci...

Watch this Scientific Journal Video about A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo at JoVE.com

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

A vascularização é essencial para abordagens de engenharia de tecidos bem sucedida. Portanto, tecnologias confiáveis ​​são necessários para avaliar o desenvolvimento de redes vasculares em tecido construções. Aqui apresentamos um método simples e de baixo custo para visualizar e quantificar a vascularização in vivo.

A pele completa Defeito Modelo Estudar a vascularização de Biomateriais<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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Research

JoVE Journal

Bioengineering

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

12.1K visualizações.  Technische Universität München.  A vascularização é essencial para abordagens de engenharia de tecidos bem sucedida. Portanto, tecnologias confiáveis ​​são necessários para avaliar o desenvolvimento de redes vasculares em tecido construções. Aqui apresentamos um método simples e de baixo custo para visualizar e quantificar a vascularização in vivo. 

Vídeo: A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

A Pele Tridimensional Humanos Reconstruir Modelo: uma Ferramenta para o Estudo da Pele Normal e Progressão Melanoma

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Bioengenharia

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Um método para avaliar pontualidade e precisão das respostas Volitional motor para vibrotátil Estímulos

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Um modelo de embrião de Zebrafish para na visualização de Vivo e Intravital análise de biomateriais-associado Staphylococcus aureus infecção

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Um modelo confiável e reprodutível tamanho crítico segmentar defeito Femoral em ratos estabilizada com um fixador externo personalizado

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

In vivo duas cores 2-Photon Imaging de células de repórter geneticamente marcadas na pele

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Modelo calvarial de aumento ósseo em coelho para avaliação do crescimento ósseo e neovascularização em materiais de substituição óssea

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...